Multilocus Variable-number Tandem-repeat Analysis Profiles Research Articles

An outbreak of Shiga toxin-producing Escherichia coli serogroup O157 linked to a lamb-feeding event.

Fifteen confirmed cases and 15 possible cases of Shiga toxin-producing Escherichia coli (STEC) O157 phage type 21/28 were linked to direct contact with lambs at a 'Lambing Live' event in the North West of England between 29 March and 21 April 2014. Twenty-one (70%) of the cases were female, 23 (77%) were children aged <16 years, of whom 14 (46%) were in the 0-5 years age group. Five children developed haemolytic uraemic syndrome. Multilocus variable number tandem repeat analysis (MLVA) profiles on 14 human cases were indistinguishable, and 6/10 animal isolates had a MLVA profile identical to the outbreak profile. Whole-genome sequencing analysis revealed that all isolates, both human and animal, fell within a 5-single nucleotide polymorphism cluster indicating the isolates belonged to the same point source. On inspection of the premises, extensive and uncontrolled physical contact between visitors and animals was occuring within the animal pens and during bottle-feeding. Public areas were visibly contaminated with animal faeces. Information to visitors, and the infection control awareness demonstrated by staff, was inadequate. Managing the risk to visitors of STEC O157 infection at animal petting events and open farms requires implementation of stringent control measures by the operator, as outlined in the industry code of practice. Enforcement action is sometimes required to prevent high-risk activities taking place at both permanent and temporary attractions.

Isolation and molecular characterization of Leptospira borgpetersenii serovar Hardjo strain Hardjobovis in the urine of naturally infected cattle in Brazil.

Most epidemiologic studies on bovine leptospirosis are based on serological tests that use antibodies against several serotypes, including the serovar Hardjo, which is widespread and considered to be the most adapted to bovine hosts. However, using only serological studies is not sufficient to identify and distinguish species of leptospires. The aim of this study was report the first isolation in Brazil of two strains serovar Hardjo obtained in urine samples from naturally infected cows in a small Brazilian dairy herd and find the genetic species and consequently the type strain Hardjobovis by molecular characterization. Fifteen dairy cows with a history of reproductive failure, such as abortion and infertility, were selected. Urine samples obtained from each animal were immediately seeded in tubes containing Ellinghausen-McCullough-Johnson-Harris culture medium. The identification of the isolates was performed by Multilocus variable-number tandem-repeat analysis (MLVA) technique and phylogenetic analysis of partial sequence of gene sec Y. From the 15 urine samples evaluated, two Leptospira were found and identified as the Londrina 49 and Londrina 54 strains. The MLVA profiles and sequencing of gene sec Y characterized the isolates as L. borgpetersenii serovar Hardjo strain Hadjobovis because it has different genetic pattern of Leptospira interrogans serovar Hardjo strain Hardjoprajitno. Therefore, more studies are needed including isolation and molecular characterization from regional strains to obtain a better knowledge about epidemiology of serovar Hardjo in bovine which may assist in future strategies of prevention and control of bovine leptospirosis.

Genetic diversity and population dynamics of Bordetella pertussis in China between 1950–2007

Pertussis is an acute respiratory infectious disease caused by the bacterium Bordetella pertussis. Although pertussis vaccination was introduced in the 1960s, pertussis is still an endemic disease in China. To better understand the genetic diversity of the Chinese B. pertussis population, we characterized 115 clinical isolates obtained in China during 1950–2007 using multilocus variable-number tandem repeat analysis (MLVA). Forty-six different B. pertussis MLVA profiles (MTs) were identified, of which 13 were new MTs. Analysis using a minimum-spanning tree showed that distinct MTs were prevalent during different periods, suggesting that a dynamic change in B. pertussis MTs occurred over time in China. The predominant MTs in recent isolates from China were different from those of many developed countries. A decreasing trend in genetic diversity of the B. pertussis population was observed following the introduction of pertussis vaccines. Similar to the pertactin 2 (prn2) allele, the novel pertussis toxin promoter (ptxP3) allele first emerged in 2000, but unlike trends elsewhere, ptxP1 remained predominant among the isolates, further reflecting the unique temporal trends in the B. pertussis population in China. Our results suggest that temporal changes in the B. pertussis population may be closely associated with vaccination coverage and the vaccine types used. These data may lead to an improved understanding of the virulence mechanism of B. pertussis and facilitate new strategies for controlling this infectious disease.

Longitudinal observational study over 38 months of verotoxigenic Escherichia coli O157:H7 status in 126 cattle herds

Verotoxigenic Escherichia coli O157:H7 (VTEC O157:H7) is an important zoonotic pathogen capable of causing infections in humans, sometimes with severe symptoms such as hemorrhagic colitis and hemolytic uremic syndrome (HUS). It has been reported that a subgroup of VTEC O157:H7, referred to as clade 8, is overrepresented among HUS cases. Cattle are considered to be the main reservoir of VTEC O157:H7 and infected animals shed the bacteria in feces without showing clinical signs of disease. The aims of the present study were: (1) to better understand how the presence of VTEC O157:H7 in the farm environment changes over an extended period of time, (2) to investigate potential risk factors for the presence of the bacteria, and (3) describe the distribution of MLVA types and specifically the occurrence of the hypervirulent strains (clade 8 strains) of VTEC O157:H7. The farm environment of 126 cattle herds in Sweden were sampled from October 2009 to December 2012 (38 months) using pooled pat and overshoe sampling. Each herd was sampled, on average, on 17 occasions (range=1–20; median=19), at intervals of 64 days (range=7–205; median=58). Verotoxigenic E. coli O157:H7 were detected on one or more occasions in 53% of the herds (n=67). In these herds, the percentage of positive sampling occasions ranged from 6% to 72% (mean=19%; median=17%). Multi-locus variable number tandem repeat analysis (MLVA) typing was performed on isolates from infected herds to identify hypervirulent strains (clade 8). Clustering of MLVA profiles yielded 35 clusters and hypervirulent strains were found in 18 herds; the same cluster was often identified on consecutive samplings and in nearby farms. Using generalized estimating equations, an association was found between the probability of detecting VTEC O157:H7 and status at the preceding sampling, season, herd size, infected neighboring farms and recent introduction of animals. This study showed that the bacteria VTEC O157:H7 were spontaneously cleared from the farm environment in most infected herds over time, and key factors were identified to prevent the spread of VTEC O157:H7 between cattle herds.

Ascertaining the relationship between Salmonella Typhimurium and Salmonella 4,[5],12:i:- by MLVA and inferring the sources of human salmonellosis due to the two serovars in Italy.

The current picture of human salmonellosis shows Salmonella Typhimurium and S. 4,[5],12:i:- as the most common serovars in Italy. The aims of this study were to investigate the genetic relationship between these serovars, as well as to test the possibility of inferring sources of human salmonellosis due to S. Typhimurium and S. 4,[5],12:i:- by using multilocus variable-number tandem repeat analysis (MLVA) subtyping data. Single isolates from 268 human sporadic cases and 325 veterinary isolates (from pig, cattle, chicken, and turkey) collected over the period 2009–2011 were typed by MLVA, and the similarities of MLVA profiles were investigated using different analytical approaches. Results showed that isolates of S. 4,[5],12:i:- were more clonal compared to S. Typhimurium and that clones of both serovars from different non-human sources were very close to those which were responsible for human infections, suggesting that source attribution by MLVA typing should be possible. However, using the Asymmetric Island Model it was not possible to obtain a confident ranking of sources responsible for human infections based on MLVA profiles. The source assignments provided by the model could have been jeopardized by the high heterogeneity found within each source and the negligible divergence between sources as well as by the limited source data available, especially for some species.

A multi-country Salmonella Enteritidis phage type 14b outbreak associated with eggs from a German producer: 'near real-time' application of whole genome sequencing and food chain investigations, United Kingdom, May to September 2014.

We report an outbreak of Salmonella Enteritidis phage type 14b (PT14b) in the United Kingdom (UK) between May and September 2014 where Public Health England launched an investigation to identify the source of infection and implement control measures. During the same period, outbreaks caused by a Salmonella Enteritidis strain with a specific multilocus variable-number tandem repeat analysis (MLVA) profile occurred in other European Union Member States. Isolates from a number of persons affected by the UK outbreak, who had initially been tested by MLVA also shared this particular profile. Cases were defined as any person infected with S. Enteritidis PT14b, resident in England or Wales and without history of travel outside of this geographical area during the incubation period, reported from 1 June 2014 onwards, with a MLVA profile of 2–11–9-7–4-3–2-8–9 or a single locus variant thereof. In total, 287 cases met the definition. Food traceback investigations in the UK and other affected European countries linked the outbreaks to chicken eggs from a German company. We undertook whole genome sequencing of isolates from UK and European cases, implicated UK premises, and German eggs: isolates were highly similar. Combined with food traceback information, this confirmed that the UK outbreak was also linked to a German producer.

Molecular epidemiology of Bordetella pertussis in the Philippines in 2012–2014

The present study was designed to determine the genotypes of circulating Bordetella pertussis in the Philippines by direct molecular typing of clinical specimens. Nasopharyngeal swabs (NPSs) were collected from 50 children hospitalized with pertussis in three hospitals during 2012-2014. Multilocus variable-number tandem repeat analysis (MLVA) was performed on the DNA extracts from NPSs. B. pertussis virulence-associated allelic genes (ptxA, prn, and fim3) and the pertussis toxin promoter, ptxP, were also investigated by DNA sequence-based typing. Twenty-six DNA extracts yielded a complete MLVA profile, which were sorted into 10 MLVA types. MLVA type 34 (MT34), which is rare in Australia, Europe, Japan, and the USA, was the predominant strain (50%). Seven MTs (MT29, MT32, MT33, and MT283-286, total 42%) were single-locus variants of MT34, while two (MT141 and MT287, total 8%) were double-locus variants of MT34. All MTs had the combination of virulence-associated allelic genes, ptxP1-ptxA1-prn1-fim3A. The B. pertussis population in the Philippines comprises genetically related strains. These strains are markedly different from those found in patients from other countries where acellular pertussis vaccines are used. The differences in vaccine types between these other countries and the Philippines, where the whole-cell vaccine is still used, may select for distinct populations of B. pertussis.

Phenotypic and Genotypic Characterization of Salmonella enterica Serovar Enteritidis Isolates Associated with a Mousse Cake-Related Outbreak of Gastroenteritis in Ningbo, China.

Salmonella enterica subsp. enterica serovar Enteritidis (Salmonella Enteritidis) is a major pathogen responsible for causing the largest number of sporadic cases and outbreaks of human salmonellosis worldwide. In this study, an outbreak of Salmonella Enteritidis involving 112 cases in Ningbo, China was investigated with a combination of genotypic subtyping methods and phenotypic analysis. The pulsed-field gel electrophoresis and multilocus variable-number tandem-repeat analysis profiles showed that most of the outbreak clinical isolates (22/23) were indistinguishable from each other and were identical to the isolates obtained from implicated mousse cakes, demonstrating that this outbreak of gastroenteritis was caused by Salmonella Enteritidis-contaminated mousse cakes. Moreover, all isolates, irrespective of source, had an identical antibiotic susceptibility pattern. Five virulence-associated genes in Salmonella pathogenicity islands and the plasmid-associated virulence genes spvB/C were present in both the food and clinical isolates. Importantly, all of these isolates can survive well under low-temperature treatment, indicating that manufacturers of foodstuffs with raw ingredients (not subjected to thermal processing) should use an effective approach to prevent or eliminate the microbial hazards to public health.

Cholera in Yangon, Myanmar, 2012-2013.

To the Editor: Vibrio cholerae O1, a causative agent of cholera, is classified into 2 biotypes, classical and El Tor. Since 1817, cholera has spread from the Indian subcontinent to other regions of the globe 7 times (1). However, little information on the occurrence of cholera and V. cholerae in Myanmar has been published. Here, we report cholera cases and characterization of 58 clinical isolates of V. cholerae O1 serotype Ogawa recovered from patients with diarrhea in Yangon, Myanmar, during February 2012–June 2013. During February and August 2012, rectal swab specimens were collected from patients suspected of having cholera in 4 hospitals in Yangon: New Yangon General Hospital, North Okkalapa General Hospital, Thingangyun Sanpya General Hospital, and Yangon Children Hospital. The specimens were cultured on thiosulfate citrate bile salts sucrose agar plates. After overnight incubation, several colonies that resembled to those of V. cholerae were confirmed as serogroup O1 by using slide agglutination tests with specific monoclonal antibodies (2). Of the tested specimens, 34 isolates carried the tcpA gene, encoding the structural subunit of toxin-coregulated pilus, and the rstR gene, the repressor gene in the cholera toxin encoding (CTX) phage (3); these results may indicate that these strains belonged to the El Tor biotype. However, identification of the sequence type of the cholera toxin B subunit gene in these isolates revealed that they were of classical type (ctxBCla). Thus, these isolates were classified as atypical El Tor V. cholerae O1 (4) carrying ctxBCla and rstREl. Currently, the predominant clones causing cholera in Asia and Africa are atypical El Tor V. cholerae, CIRS101, and CIRS101-like variants (5,6). Myanmar isolates from 2012 and the CIRS101 strain contained a single nucleotide polymorphism in the tcpA gene at nt 266 (A→G) of the prototype seventh pandemic El Tor (N16961) strain. Pulsed-field gel electrophoresis (PFGE) (7) using the 2012 isolates revealed 9 patterns (Table). During the initial phase of cholera occurrences, V. cholerae O1 was mainly isolated from adults, and 9 pulsotypes were observed. During the later period (May–August), most isolates were from children <5 years of age, and pulsotype Y6 predominated. Table Characterization of Vibrio cholerae O1 isolates from patients in Myanmar, 2012–2013* We carried out multilocus variable-number tandem-repeat analysis (MLVA) (8) of the 2012 isolates to resolve distinct populations. MLVA yielded 13 isolate types, and all 18 isolates of pulsotype Y6 exhibited either MLVA profile 11.6.6.17.17 or a closely related profile that differed only by 1 repeat number. These data suggest that cholera was contracted mainly in adults and was caused by multiclonal V. cholerae O1. However, in children, V. cholerae has transformed from single clonal expansion since May 2012. During March–June 2013, we extended our studies to characterize cholera organisms isolated from patients with severe diarrhea who were admitted to the original 4 hospitals as well as 2 additional hospitals, Yankin Children Hospital and Insein General Hospital. Of 24 cases, 16 patients showed symptoms of severe dehydration, including 1 patient who experienced shock. Other common symptoms in this patient population included fever (50%, 12/24), vomiting (92%, 22/24), and abdominal pain (33%, 8/24). Although fever is less common among patients with cholera-associated diarrhea (9), the frequency of fever was considerably high in this study. PFGE reveled 23 of the 24 isolates were identical to pulsotype Y7, which was the second-most prevalent pattern in 2012; MLVA profiles were also similar to those from 2012. Thus, the occurrences of cholera in 2013 may have been related to persistent transmission of a clone from 2012. According to surveillance records from the Yangon Regional Health Center, the reported number of diarrhea cases in Yangon increased from 11,651 in 2010 and 11,016 in 2011 to 15,540 in 2012 and 13,919 in 2013. Although there were no reports of cholera outbreaks in Yangon, PFGE/MLVA results revealed that most of the cholera cases in this study were caused by isolates belonging to identical or closely related types. Thus, cholera outbreaks could have occurred in Yangon, and the related clone may have persisted. In Myanmar, the illness rate for severe diarrhea is estimated to be 2.6–3.5 per 100,000 persons and the mortality rate is 0.04–0.1 per 100,000 (10). In this study, the detection rates of V. cholerae O1 in stools from patients with severe diarrhea were 23% (49/213 cases) in 2012 and 14% (35/250 cases) in 2013, respectively. Although our investigation is merely the tip of the iceberg for studies of cholera in Myanmar, our data provide crucial initial insights into the genetic backgrounds of recent Yangon isolates of V. cholerae O1. Epidemiologic surveillance linked to laboratory investigations is need to minimize the risk for V. cholerae infection in children.

Molecular typing of Escherichia coli O157:H7 isolates from Swedish cattle and human cases: population dynamics and virulence.

While all verotoxin-producing Escherichia coli O157:H7 bacteria are considered potential pathogens, their genetic subtypes appear to differ in their levels of virulence. The aim of this study was to compare the distribution of subtypes of E. coli O157:H7 in the cattle reservoir and in human cases with and without severe complications in order to gain clues about the relationship between subtype and relative virulence. A lineage-specific polymorphism assay (LSPA-6), multilocus variable-number tandem-repeat analysis (MLVA), and a novel real-time PCR assay to identify clade 8 were applied to a large and representative set of isolates from cattle from 1996 to 2009 (n = 381) and human cases from 2008 to 2011 (n = 197) in Sweden. Draft genome sequences were produced for four selected isolates. The E. coli O157:H7 isolates in Swedish cattle generally belonged to four groups with the LSPA-6 profiles 211111 (clade 8/non-clade 8), 213111, and 223323. The subtype composition of the cattle isolates changed dramatically during the study period with the introduction and rapid spread of the low-virulence 223323 subtype. The human cases presumed to have been infected within the country predominantly carried isolates with the profiles 211111 (clade 8) and 213111. Cases progressing to hemolytic-uremic syndrome (HUS) were mostly caused by clade 8, with MLVA profiles consistent with Swedish cattle as the source. In contrast, infections contracted abroad were caused by diverse subtypes, some of which were associated with a particular region. The work presented here confirms the high risk posed by the clade 8 variant of E. coli O157:H7. It also highlights the dynamic nature of the E. coli O157:H7 subtype composition in animal reservoirs and the importance of this composition for the human burden of disease.

Molecular epidemiology of the pertussis epidemic in Washington State in 2012.

Although pertussis disease is vaccine preventable, Washington State experienced a substantial rise in pertussis incidence beginning in 2011. By June 2012, the reported cases reached 2,520 (37.5 cases per 100,000 residents), a 1,300% increase compared with the same period in 2011. We assessed the molecular epidemiology of this statewide epidemic using 240 isolates collected from case patients reported from 19 of 39 Washington counties during 2012 to 2013. The typing methods included pulsed-field gel electrophoresis (PFGE), multilocus variable number tandem repeat analysis (MLVA), multilocus sequence typing (MLST), and pertactin gene (prn) mutational analysis. Using the scheme PFGE-MLVA-MLST-prn mutations-Prn deficiency, the 240 isolates comprised 65 distinct typing profiles. Thirty-one PFGE types were found, with the most common types, CDC013 (n = 51), CDC237 (n = 44), and CDC002 (n = 42), accounting for 57% of them. Eleven MLVA types were observed, mainly comprising type 27 (n = 183, 76%). Seven MLST types were identified, with the majority of the isolates typing as prn2-ptxP3-ptxA1-fim3-1 (n = 157, 65%). Four different prn mutations accounted for the 76% of isolates exhibiting pertactin deficiency. PFGE provided the highest discriminatory power (D = 0.87) and was found to be a more powerful typing method than MLVA and MLST combined (D = 0.67). This study provides evidence for the continued predominance of MLVA 27 and prn2-ptxP3-ptxA1 alleles, along with the reemergence of the fim3-1 allele. Our results indicate that the Bordetella pertussis population causing this epidemic was diverse, with a few molecular types predominating. The PFGE, MLVA, and MLST profiles were consistent with the predominate types circulating in the United States and other countries. For prn, several mutations were present in multiple molecular types.

Clonal distribution of monophasic Salmonella enterica subsp. enterica serotype 4,[5],12:i:- in the Czech Republic.

Abstract Salmonella 4,[5],12:i:- has become the third most common serotype in Europe, including the Czech Republic. In this study, phenotypic and genotypic methods for a more detailed description of this serotype were used. Analysis of a limited number of isolates revealed that 76% of them belonged to phage type DT193. Also, rare phage types DT208 and U311 were identified. In total, 88.6% of the isolates were resistant to at least four antimicrobial agents. In this study, 24 multilocus variable-number tandem-repeat analysis profiles were detected, and some of them matched with the profiles recently described in Europe.

Molecular characterization of foodborne-associated Staphylococcus aureus strains isolated in Shijiazhuang, China, from 2010 to 2012

Staphylococcus aureus is an opportunistic pathogen commonly identified from food poisoning–associated foodstuffs. From 1996 to the present, S. aureus isolates have been found to exhibit increasing resistance to antimicrobial drugs. The aim of this study was to assess the molecular epidemiology properties of various S. aureus isolates through molecular typing and to investigate their characterization based on their production of enterotoxins and hemolysins and their resistance to antibiotics. A total of 78 coagulase-positive staphylococcal strains isolated from food or clinical samples were analyzed. Eight VNTR loci were used to genotype the 78 isolates, and this analysis resulted in 39 different multilocus variable number tandem repeat analysis (MLVA) profiles. The isolates recovered from a single outbreak exhibited the same MLVA profile. According to CLSI, 97.4% of the isolates were resistant to penicillin, whereas only 3.8% were methicillin-resistant Staphylococcus aureus strains. Through multiplex PCR, 87.2% of the isolates were shown to be enterotoxigenic (SEs), and the most common genes present were sea, sem, seg, seu, and sek. In conclusion, this study demonstrates the prevalence of staphylococcal enterotoxins, the contents of virulent factors, and the characteristics of β-lactam antibiotic resistance in 78 S. aureus isolates. These findings emphasize the need to prevent the presence of S. aureus strains and SE production in foods. Our results also demonstrate that MLVA is a useful and powerful method for epidemiological studies of S. aureus. In contrast to multilocus sequence typing, the MLVA method is a simpler and more rapid method for epidemiological typing with a higher discriminatory power.

Multilocus variable number tandem repeat analysis of Edwardsiella piscicida isolates pathogenic to fish.

This study describes a novel multilocus variable number tandem repeat analysis (MLVA) based on six variable number of tandem repeat (VNTR) loci for genotyping of 37 Edwardsiella piscicida (previously Edwardsiella tarda) isolates from multiple sources. The number of alleles identified for each of the six VNTR loci ranged from 3 to 5 with VNTR loci 1 (DI = 0.632) and 3 (DI = 0.644), displaying the highest degrees of polymorphism. MLVA typing of the 37 E. piscicida isolates resulted in the identification of five major clusters consistent with their geographical origins, and were designated as MLVA types I, II, III, IV and V. Types III and V were resolved further into subtypes largely consistent with outbreak source. An MLVA profile comprising a string of integers representing the number of tandem repeats for each allele provided a unique identification for each MLVA type and/or strain. The MLVA protocol described in the current study is robust, relatively simple, has a higher power of resolution than multilocus sequence analysis (MLSA) and is capable of discriminating closely related isolates.

Genotyping of outbreak-associated and sporadic Yersinia pseudotuberculosis strains by novel multilocus variable-number tandem repeat analysis (MLVA)

Yersinia pseudotuberculosis human infections caused by serotype O:1 and O:3 isolates have been common in Finland and have also caused outbreaks. Epidemiological studies on the outbreaks have been limited by the lack of accurate typing methods. During the recent years, multilocus variable-number tandem repeat analysis (MLVA) has been successfully applied for molecular typing of several bacterial pathogens. We designed an MLVA scheme based on seven loci for Y. pseudotuberculosis. The method was able to discriminate clinical isolates of serotypes O:1 and O:3 into several MLVA types. The MLVA profiles were based on the number of 6 to 9bp long tandem repeats in each locus. The number of repeats varied from 1 to 23 depending on the locus. The loci were all located in the bacterial chromosome for stability of the markers. The MLVA method developed was serotype-specific and will be a new additional tool for the epidemiological investigations of isolates associated with disease outbreaks and for comparison of sporadic isolates.

Application of multilocus variable number tandem repeat analysis to monitor Verocytotoxin-producingEscherichia coliO157 phage type 8 in England and Wales: emergence of a profile associated with a national outbreak

Evaluation of multilocus variable number tandem repeat analysis (MLVA) to subtype all isolates of Vero cytotoxin-producing Escherichia coli O157 phage type 8 in England and Wales. Over a 13 month period from December 2010, 483 isolates of VTEC O157 PT8 were tested by MLVA; 39% were received in the first 4months of 2011, when infections are generally low. One profile, or single locus variants of it, was present in 249 (52%) isolates but was not common previously. These cases represented a national increase in PT8, associated epidemiologically with soil-contaminated vegetables. Most of the 177 other MLVA profiles were unique to a single isolate. Profiles shared by >1 isolate included cases from two small community, food-borne outbreaks and 11 households. Several shared profiles were found among 23 isolates without known links. Apart from one group, isolates linked to travel abroad had very diverse profiles. Multilocus variable number tandem repeat analysis discriminated apparent sporadic isolates of the same PT and assisted in detection of cases in an emerging national outbreak. Multilocus variable number tandem repeat analysis is an epidemiologically valid complement to surveillance and applicable as a rapid, practical test for large numbers of isolates.

Characterization ofSalmonella entericaSubsp.entericaSerovar 4,[5],12:i:- Clones Isolated from Human and Other Sources in Switzerland Between 2007 and 2011

Salmonella enterica subsp. enterica serovar 4,[5],12:i:- is a monophasic variant of Salmonella Typhimurium. In this study, a total of 651 human and 107 food and environmental isolates of serovar 4,[5],12:i:- recovered from 2007 through 2011 in Switzerland were characterized by antibiotic resistance profiles and pulsed-field gel electrophoresis (PFGE). In addition, a selection of isolates belonging to the most frequent PFGE patterns was further subjected to multilocus variable-number tandem-repeat analysis (MLVA) and phage typing. Over the years 2007-2011, the reports of salmonellosis caused by Salmonella enterica serovar 4,[5],12:i:- significantly increased. A high prevalence of multidrug-resistant isolates, mainly showing an ampicillin-streptomycin-sulfonamide-tetracycline resistance pattern (ASSuT), was observed. In addition, four extended spectrum beta lactamase (ESBL) (CTX-M-55)-producing isolates were found. XbaI PFGE analysis of all isolates revealed over 150 different pulsotypes, and generally showed a considerable diversity within the monophasic isolates. Nevertheless, among these we identified seven dominant profiles, which encompassed 66% of all isolates tested. The PFGE type STYMXB.0131 dominated among human as well as food isolates. Multilocus variable-number tandem-repeat analysis profile 3-12-10-0-0211, which, in many cases, coincided with PFGE type STYMXB.0131 and phage type DT193 were the most prevalent types found for the isolates further characterized by these typing methods. Our data provide strong evidence for a spread of two specific Salmonella serovar 4,[5],12:i:- clones (PFGE pattern STYMXB.0131, resistance type ASSuT) and (PFGE pattern STYMXB.0131, resistance type SSuT). In contrast to the human isolates, the pork/poultry isolates expressed predominantly the SSuT resistance type.

Prolonged outbreak of meticillin-resistant Staphylococcus aureus in a cardiac surgery unit linked to a single colonized healthcare worker

In low- as well as in high-prevalence settings, healthcare workers (HCWs) may be a substantial, under-recognized, reservoir of meticillin-resistant Staphylococcus aureus (MRSA) and an important potential source of transmission to patients. To report an outbreak of MRSA in a cardiac surgery unit in England over a 10-month period. Cases were defined as patients and staff on the cardiac surgery unit from whom the outbreak strain was newly isolated between 20 May 2011 and 16 March 2012. Representative isolates from all cases were characterized by spa-typing, pulsed-field gel electrophoresis and multi-locus variable-number tandem-repeat analysis (MLVA). Four patients appeared to acquire MRSA during their inpatient stay on the cardiac surgery unit. All four patients and one HCW were found to be carrying an identical epidemic (E)MRSA-15 strain (spa t032, pulsotype A, MLVA profile 16-6-3-1-1-17-1-4). No other members of staff were found to be colonized with MRSA. The colonized HCW was thought to be the source of the outbreak and was decolonized using a combination of nasal mupirocin, chlorhexidine body wash and oral rifampicin and doxycycline. This report highlights recent changes in the epidemiology of MRSA in England and suggests an important role for colonized HCWs in the transmission of MRSA to patients. Screening HCWs may provide an increasingly valuable strategy in managing linked hospital acquisitions and well-defined outbreaks where initial investigation does not reveal a source.

The population structure of Salmonella enterica Enteritidis in Iran analyzed by multiple-locus variable-number tandem repeat analysis

Salmonella enterica Enteritidis is the most frequent etiological agent of salmonellosis in humans and poultry. To understand the genetic diversity of S. Enteritidis in Iran, we examined 69 chicken isolates from 18 broiler farms and six non-epidemic human isolates from six geographically distant provinces by multi-locus variable-number tandem repeat analysis (MLVA). Among SE2, SE3, SE5, SE7, SE8, SENTR4, and SENTR7, only SE5 with four and SENTR7 with two alleles, respectively, proved variable giving estimates of locus genetic diversity of 0.58 and 0. In all, six closely related MLVA profiles were identified among which three were commonly represented by human and chicken isolates. This population homogeneity contrasts with the high diversity at these loci reported elsewhere and is likely a consequence of a single clone of S. Enteritidis distributed across Iran.

Direct molecular typing of Bordetella pertussis from clinical specimens submitted for diagnostic quantitative (real-time) PCR

Molecular typing of Bordetella pertussis is routinely performed on bacterial isolates, but not on DNA extracted from nasopharyngeal aspirates or pernasal swabs submitted for diagnostic real-time PCR (qPCR). We investigated whether these DNA extracts were suitable for multilocus variable-number tandem repeat analysis (MLVA) and DNA sequence-based typing. We analysed all the available qPCR-positive samples received by our laboratory from patients <1 year of age between January 2008 and August 2010. Eighty-one per cent (106/131) of these generated a complete MLVA profile. This rose to 92 % (105/114) if only samples positive for both of the two targets used for the B. pertussis PCR (insertion element IS481 and pertussis toxin promoter ptxP) were analysed. Sequence-based typing of the pertactin, pertussis toxin S1 subunit and pertussis promoter regions (prn, ptxA and ptxP) was attempted on 89 of the DNA extracts that had generated a full MLVA profile. Eighty-three (93 %) of these produced complete sequences for all three targets. Comparison of molecular typing data from the 89 extracts with those from 111 contemporary bacterial isolates showed that the two sources yielded the same picture of the B. pertussis population [dominated by the MLVA-27 prn(2) ptxA(1) ptxP(3) clonal type]. There was no significant difference in MLVA type distribution or diversity between the two sample sets. This suggests that clinical extracts can be used in place of, or to complement, bacterial cultures for typing purposes (at least, in this age group). With small modifications to methodology, generating MLVA and sequence-based typing data from qPCR-positive clinical DNA extracts is likely to generate a complete dataset in the majority of samples from the <1 year age group. Its success with samples from older subjects remains to be seen. However, our data suggest that it is suitable for inclusion in molecular epidemiological studies of the B. pertussis population or as a tool in outbreak investigations.